Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

• Published in: Methods (San Diego, Calif.) Vol. 57; no. 4; pp. 473 - 485
• Main Authors: Marshall, Christopher B., Meiri, David, Smith, Matthew J., Mazhab-Jafari, Mohammad T., Gasmi-Seabrook, Geneviève M.C., Rottapel, Robert, Stambolic, Vuk, Ikura, Mitsuhiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2012
• Subjects: Animals; Cell Extracts - chemistry; Dyneins - chemistry; Enzyme Assays; Fluorescent Dyes - chemistry; GTP Phosphohydrolases - chemistry; GTPase activating proteins (GAPs); GTPase-Activating Proteins - chemistry; Guanine nucleotide exchange factors (GEFs); Guanine Nucleotide Exchange Factors - chemistry; Guanosine Triphosphate - chemistry; HEK293 Cells; Humans; Hydrolysis; Lfc/GEF-H1; Magnetic Resonance Spectroscopy; Monomeric GTP-Binding Proteins - chemistry; Neuropeptides - chemistry; Ras Homolog Enriched in Brain Protein; Real-time NMR; Recombinant Proteins - chemistry; Signal Transduction; Small GTPase cycle; Transfection; Tuberous sclerosis complex (TSC); Tumor Suppressor Proteins - chemistry; Real-time NMR; GTPase activating proteins (GAPs); Tuberous sclerosis complex (TSC); Lfc/GEF-H1; Small GTPase cycle; Guanine nucleotide exchange factors (GEFs)
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2012.06.014

[Display omitted] . ► NMR is used to directly monitor activation state of GTPase proteins in real time. ► Application to intrinsic nucleotide hydrolysis/exchange as well as GAP/GEF assays. ► Unambiguous readout enables assays of GAP/GEF activities in mammalian cell lysates. ► Small GTPases Ras, RhoA, and Rheb are presented examples. ► Activities of TSC2, p120 RasGAP, Sos, PDZ-RhoGEF and Lfc/GEF-H1 were assayed. The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.